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Proteolysis-a characteristic of tumor-initiating cells in murine metastatic breast cancer.

ABSTRACT: Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The cell population, characterized by the marker profile CD24+CD90+CD45-, showed a high tumorigenicity compared to non-CD24+CD90+CD45- cancer cells in colony formation assays, as well as upon orthotopic transplantation into the mammary fat pad of mice. In addition, these orthotopically grown CD24+CD90+CD45- TICs metastasized to the lungs. The transcriptome of TICs freshly isolated from primary tumors by cell sorting was compared with that of sorted non-CD24+CD90+CD45- cancer cells by RNA-seq. In addition to more established TIC signatures, such as epithelial-to-mesenchymal transition or mitogen signaling, an upregulated gene set comprising several classes of proteolytic enzymes was uncovered in the TICs. Accordingly, TICs showed high intra- and extracellular proteolytic activity. Application of a broad range of protease inhibitors to TICs in a colony formation assay reduced anchorage independent growth and had an impact on colony morphology in 3D cell culture assays. We conclude that CD24+CD90+CD45- cells of the MMTV- PyMT mouse model possess an upregulated proteolytic signature which could very well represent a functional hallmark of metastatic TICs from mammary carcinomas.

SUBMITTER: Hillebrand LE

PROVIDER: S-EPMC5295428 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Proteolysis-a characteristic of tumor-initiating cells in murine metastatic breast cancer.

Hillebrand Larissa E LE Bengsch Fee F Hochrein Jochen J Hülsdünker Jan J Bender Julia J Follo Marie M Busch Hauke H Boerries Melanie M Reinheckel Thomas T

Oncotarget 20160901 36

Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The ...[more]

PMID: 27542270

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Project description:PurposeEvidence is lacking whether the number of breast tumor-initiating cells (BT-ICs) directly correlates with the sensitivity of breast tumors to chemotherapy. Here, we evaluated the association between proportion of BT-ICs and chemoresistance of the tumors.MethodsImmunohistochemical staining(IHC) was used to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and proliferating cell nuclear antigen, and TUNEL was used to detect the apoptosis index. The significance of various variables in patient survival was analyzed using a Cox proportional hazards model. The percentage of BT-ICs in breast cancer cell lines and primary breast tumors was determined by ALDH1 enzymatic assay, CD44(+)/CD24(-) phenotype and mammosphere formation assay.ResultsALDH1 expression determined by IHC in primary breast cancers was associated with poor clinical response to neoadjuvant chemotherapy and reduced survival in breast cancer patients. Breast tumors that contained higher proportion of BT-ICs with CD44(+)/CD24(-) phenotype, ALDH1 enzymatic activity and sphere forming capacity were more resistant to neoadjuvant chemotherapy. Chemoresistant cell lines AdrR/MCF-7 and SK-3rd, had increased number of cells with sphere forming capacity, CD44(+)/CD24(-) phenotype and side-population. Regardless the proportion of T-ICs, FACS-sorted CD44(+)/CD24(-) cells that derived from primary tumors or breast cancer lines were about 10-60 fold more resistant to chemotherapy relative to the non- CD44(+)/CD24(-) cells and their parental cells. Furthermore, our data demonstrated that MDR1 (multidrug resistance 1) and ABCG2 (ATP-binding cassette sub-family G member 2) were upregulated in CD44(+)/CD24(-) cells. Treatment with lapatinib or salinomycin reduced the proportion of BT-ICs by nearly 50 fold, and thus enhanced the sensitivity of breast cancer cells to chemotherapy by around 30 fold.ConclusionsThese data suggest that the proportion of BT-ICs is associated with chemotherapeutic resistance of breast cancer. It highlights the importance of targeting T-ICs, rather than eliminating the bulk of rapidly dividing and terminally differentiated cells, in novel anti-cancer strategies.

Project description:Obesity is a risk factor for triple-negative breast cancer (TNBC) incidence and poor outcomes, but the underlying molecular biology remains unknown. We previously identified in TNBC cell cultures that expression of epigenetic reader methyl-CpG-binding domain protein 2 (MBD2), specifically the alternative mRNA splicing variant MBD variant 2 (MBD2_v2), is dependent on reactive oxygen species (ROS) and is crucial for maintenance and expansion of cancer stem cell-like cells (CSCs). Because obesity is coupled with inflammation and ROS, we hypothesized that obesity can fuel an increase in MBD2_v2 expression to promote the tumor-initiating CSC phenotype in TNBC cells in vivo. Analysis of TNBC patient datasets revealed associations between high tumor MBD2_v2 expression and high relapse rates and high body mass index (BMI). Stable gene knockdown/overexpression methods were applied to TNBC cell lines to elucidate that MBD2_v2 expression is governed by ROS-dependent expression of serine- and arginine-rich splicing factor 2 (SRSF2). We employed a diet-induced obesity (DIO) mouse model that mimics human obesity to investigate whether obesity causes increased MBD2_v2 expression and increased tumor initiation capacity in inoculated TNBC cell lines. MBD2_v2 and SRSF2 levels were increased in TNBC cell line-derived tumors that formed more frequently in DIO mice relative to tumors in lean control mice. Stable MBD2_v2 overexpression increased the CSC fraction in culture and increased TNBC cell line tumor initiation capacity in vivo. SRSF2 knockdown resulted in decreased MBD2_v2 expression, decreased CSCs in TNBC cell cultures, and hindered tumor formation in vivo. This report describes evidence to support the conclusion that MBD2_v2 expression is induced by obesity and drives TNBC cell tumorigenicity, and thus provides molecular insights into support of the epidemiological evidence that obesity is a risk factor for TNBC. The majority of TNBC patients are obese and rising obesity rates threaten to further increase the burden of obesity-linked cancers, which reinforces the relevance of this report.

Dataset Information

Proteolysis-a characteristic of tumor-initiating cells in murine metastatic breast cancer.

Publications

Proteolysis-a characteristic of tumor-initiating cells in murine metastatic breast cancer.

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